Nitric oxide (NO) achieves its biological functions through a balance between its synthesis and inactivation. The biosynthesis of NO is highly regulated and well documented, whereas its inactivation is much less understood. The major pathways for NO inactivation include reactions with oxygenated hemoglobin [HbFe (ll) O2] and various free radicals. Conventional wisdom suggests that NO inactivation by HbFe (ll) O2 is not regulated, since NO is thought to diffuse freely and rapidly across the red blood cell (RBC) membrane. Recent findings have shown that NO transport into RBCs is controlled by the membrane skeleton proteins. The rate of NO consumption by RBCs can be modulated by perturbing the cytoskeleton network through cytoskeleton binding proteins such as Band 3. In particular. formation of iron-nitrosyl-hemoglobin [HbFe (ll) NO] (-0.1%) increased the NO consumption rate. This regulator is of physiological and pathological importance as HbFe (ll) NO is formed during hypoxia and has been detected in humans under various conditions. The purpose of this application is thus to investigate the biochemical mechanisms underlying the nitrosylHb-mediated regulations and to determine their physiological/pathological roles. It is hypothesized that HbFe (ll) NO in the "super T" state binds to Band 3 and shifts its population to the dimer form, which loosens the cytoskeleton network. Since HbFe (ll) NO may be produced in the lungs under hypoxia, the HbFe (ll) NO regulated NO consumption may participate in hypoxic pulmonary vasoconstriction. Moreover, based on preliminary data, it is further hypothesized that HbFe (ll) NO attenuates the NO-mediated coronary vasodilation. Specific aim 1 will investigate the mechanisms involved in this regulation using biochemical and biophysical techniques which probe the state of cytoskeleton and Band 3 protein. Specific aim 2 will focus on the functional roles of this regulation using isolated porcine pulmonary and coronary microvessels. Together, these results will suggest clinical relevance and potential interventions.